Therapies have been developed to treat a wide range of formerly intractable diseases or conditions, such as multiple sclerosis; various cancers, autoimmune diseases such as Crohn's disease, ulcerative colitis, psoriasis, psoriatic arthritis, ankylosing spondylitis, and rheumatoid arthritis; and organ transplant rejection. A partial list of some of these therapies, and their mechanisms of action, is shown in TABLE 1. It can be seen, from TABLE 1, that these therapies cause immunosuppression either by inactivation, inhibition, or immobilization of immune effector cells (B-cells, T-cells, dendritic cells, monocytes, macrophages), or by cytotoxic side effects on immune effector cells.
TABLE 1Drugs that have been shown to trigger JC Virus and result in PML:DrugTreatmentMOAIMMUNOMODULATORSBrentuximab vedotinHodgkin's lymphomasanti-CD30RituximabB-cell cancersinhibits B-cell activityNatalizumabMultiple Sclerosis and Crohn'santi-alpha-4 integrin. α4-integrin isDiseaserequired for white blood cells tomove into organs by preventingtheir crossing of blood vessel wallsto reach affected organsFingolimodMultiple SclerosisEfalizumabPsoriasisinhibits lymphocyte activationVedolizumabulcerative colitis and Crohn'sBlocking the α4β7 integrindiseasecauses gut-selective anti-inflammatory activityDimethyl fumaratepsoriasis, necrobiosis lipoidica,hypoxic cell radiosensitizergranuloma annulare,sarcoidosis, and MultipleSclerosisIMMUNOSUPPRESSANTSBelataceptimmunosuppressantblocks T-cell activationTacrolimusimmunosuppressantCalcineurin Inhibitors/T-cellinhibitorsSirolimusimmunosuppressantmTOR inhibitorsGlucocorticoidsimmunosuppressantsteroidsMethotrexateimmunosuppressantantimetabolitesAzathioprineimmunosuppressantantimetabolitesCyclosporineimmunosuppressantT-cell inhibitorsCyclophosphamideimmunosuppressantalkylating agentsChlorambucilimmunosuppressantalkylating agentsMycophenolate mofetilimmunosuppressantAntiproliferative/antibioticagentDaclizumabimmunosuppressantprevents T-cell activationInfliximabCrohn's disease, ulcerativeanti-TNFαcolitis, psoriasis, psoriaticarthritis, ankylosing spondylitis,and rheumatoid arthritisOcrelizumabImmunosuppressantHumanized anti-CD20monoclonal antibody that bindsCD20 on B-lymphocytesAlemtuzumabImmunosuppressantAn anti-CD52 monoclonalantibody that binds CD52, onthe surface ofmature lymphocytes to treatchronic lymphocytic leukemia,cutaneous T-cell lymphoma, T-cell lymphoma and MultipleSclerosisLaquinimodImmunomodulatorFor treatment of MSDaclizumabImmunosuppressantBinds to CD25, the alpha subunitof the IL-2 receptor of T-cells. Ahumanized anti-CD25monoclonal antibody for thetreatment of relapsing forms ofMS.
The immunosuppressive action of these therapies carries the risk of activation of opportunistic pathogens that are normally kept in check by the immune system. Among the most serious risks is the risk of activation of John Cunningham Virus (JCV), a human neurotropic polyomavirus. JCV is the etiological agent of a fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). Lytic infection of JCV in glial cells of the central nervous system (CNS) results in the death of oligodendrocytes, the cells that are responsible for the production of myelin sheaths of neurons in the brain. This leads to a broad range of mild to severe neurological disturbances and eventually death (Berger, 2011). There are a number of predisposing factors to PML, all of which involve some level of impairment of the immune system.
Seroepidemological data indicate that the 75-80% of the human population is infected with JCV. Much of this infection occurs during childhood, by largely unknown routes (Saribas, et al., 2010). The virus typically remains latent, causing no symptoms. In a setting of impaired immunity, especially cellular immunity, the virus can reactivate, proliferating and inducing the symptoms of PML (Waggoner, et al, 2009). Latent virus can be maintained in the urinary tract and bone marrow, in the spleen and other lymphoid tissues, and in the CNS (Bayliss, et al., 2012). Reactivation during immunosuppression can reflect the reactivation of latent virus in the CNS, as well as the hematogenous spread of reactivated virus to the CNS (Bag, et al., 2010).
The JCV genome is comprised of double-stranded circular DNA of 5.1 kb in size, which codes for two classes of proteins at the early phase of viral infection, i.e. before DNA replication, and at the late phase of the infection cycle (DeCaprio, et al., 2013). A bi-directional coding sequence positioned between the early and late genes is responsible for viral gene expression and contains the origin of viral DNA replication. The viral early protein, large T-antigen (T-Ag), and a family of smaller sized T-Ag proteins, are produced by alternative splicing, and have a regulatory role in orchestrating the viral replication cycle. The large T-Ag, in particular, is responsible for initiation of viral DNA replication and the stimulation of viral late gene transcription, and thus is critical for all aspects of the viral life cycle (for review see White and Khalili, 2004). T-Ag binds to several cellular proteins such as p53 and pRb, and dysregulates proliferation of host cells. The late proteins include the viral capsid proteins VP1, VP2, and VP3 and a small regulatory protein known as agnoprotein (Khalili, et al., 2005).
Treatments for autoimmune disorders such as multiple sclerosis and rheumatoid arthritis, with new therapeutic immunomodulatory monoclonal antibodies, including natalizumab (Chakley and Berger, 2013) efalizumab (Schwab, et al., 2012), and rituximab Clifford, et al., 2011), are recognized as a predisposing factors for PML (Nagayama, et al., 2013). As a consequence of the risk of JCV activation and PML, these treatments, and many of the other treatments listed in Table 1, must to be administered in sub-optimal concentrations with extensive patient monitoring. In some cases, the PML risk is sufficient to cause the removal of immunosuppressive drugs from the market, thereby barring patient access to potentially life-saving treatments.
A number of treatment options have been applied to PML, largely without success (Tavazzi, et al. 2012). Diverse approaches have targeted various points in the viral life cycle, such as cellular entry and replication. Since interaction between JCV and the serotonin 2A receptor (5-HT2AR) has been reported to be required for viral entry (Elphick, et al., 2004), risperidone, which binds 5HT2AR, has been tested but found to have no effect (Chapagain, et al., 2008). Small molecule inhibitors of viral replication such as cidofovir have been tested in vitro and in vivo, but have yielded conflicting results (Andrei, et al., 1997, Hou and Major, 1998). Alternative strategies are urgently required for dealing with this fatal demyelinating disease.
One potentially effective strategy would be to eliminate latent JCV from the host cells of patients prior to the start of immunosuppressive therapy, or during and after the course of therapy. With no latent virus to be activated, there would be no need to treat an active JCV infection. New and developing gene editing systems that target the JCV viral genome would be particularly attractive tools for JCV elimination. Example systems include zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and Clustered Regulatory Interspaced Short Palindromic Repeat (CRISPR)-associated nuclease systems (Gaj, et al., 2013).
In particular, tools and techniques based on CRISPR/endonuclease DNA editing systems offer unprecedented control over genome editing (Mali, et al., 2013, Hsu, et al., 2014). The CRISPR/Cas9 (CRISPR-associated endonuclease 9) system was developed from the adaptive immune system of bacteria and archaea. The CRISPR/Cas9 system uses short guide RNAs (gRNAs) to direct the cleavage of specific nucleic acid target sequences by a Cas9 endonuclease (Bhaya, et al., 2011). The cleavage, usually a blunt ended double-strand cut, can cause deletions, insertions, and excisions of stretches of DNA, caused by defective DNA repair. Recently, it was reported that CRISPR/Cas9 can be used to eliminate JCV from latently infected cells and prevent new JCV infection (Wollebo, et al., 2015). Recently, the range of targets has been expanded by the introduction of a CRISPR system that utilizes an alternative endonuclease, Cpf1, which is directed by gRNAs different from those which direct Cas9, to target sequences different from those cleaved by Cas9 (Zetsche, et al., 2015). There is a need for compositions and methods for the employment of these gene editing systems in treatments to eliminate latent JCV from patient cells prior to immunosuppressive treatments.